2 macrophages. selleck chemicals llc Microbiology 2007,153(Pt 6):1756–1771.PubMedCrossRef 118. Joseph B, Przybilla K, Stuhler C, Schauer K, Slaghuis J, Fuchs TM, Goebel W: Identification of Listeria monocytogenes genes contributing to intracellular replication by expression profiling and mutant screening. J Bacteriol 2006,188(2):556–568.PubMedCrossRef 119. Stojiljkovic I, Baumler AJ, Heffron F: Ethanolamine utilization in Salmonella typhimurium : nucleotide sequence, protein expression, and mutational analysis of the cchA cchB eutE eutJ

eutG eutH gene cluster. J Bacteriol 1995,177(5):1357–1366.PubMed 120. Dibden DP, Green J: In vivo cycling of the Escherichia coli transcription factor FNR between active and inactive

states. Microbiology 2005,151(Pt 12):4063–4070.PubMedCrossRef 121. Jones see more HM, Gunsalus RP: Regulation of Escherichia coli fumarate reductase ( frdABCD ) operon expression by respiratory electron acceptors and the fnr gene product. J Bacteriol 1987,169(7):3340–3349.PubMed 122. Melville SB, Gunsalus RP: Mutations in fnr that alter anaerobic regulation of electron SBI-0206965 nmr transport-associated genes in Escherichia coli . J Biol Chem 1990,265(31):18733–18736.PubMed 123. McCrindle SL, Kappler U, McEwan AG: Microbial dimethylsulfoxide and trimethylamine-N-oxide respiration. Adv Microb Physiol 2005, 50:147–198.PubMedCrossRef 124. Privalle CT, Fridovich I: Transcriptional and maturational effects of manganese and iron on the biosynthesis of manganese-superoxide dismutase in Escherichia coli . J Biol Chem 1992,267(13):9140–9145.PubMed 125. McCollister BD, Bourret TJ, Gill R, Jones-Carson J, Vazquez-Torres A: before Repression of SPI2 transcription by nitric oxide-producing, IFNgamma-activated macrophages promotes maturation of Salmonella phagosomes. J Exp Med 2005,202(5):625–635.PubMedCrossRef 126. Foster JW, Bearson B: Acid-sensitive mutants of Salmonella typhimurium identified through a dinitrophenol lethal screening strategy. J Bacteriol 1994,176(9):2596–2602.PubMed

127. Pettis GS, Brickman TJ, McIntosh MA: Transcriptional mapping and nucleotide sequence of the Escherichia coli fepA-fes enterobactin region. Identification of a unique iron-regulated bidirectional promoter. J Biol Chem 1988,263(35):18857–18863.PubMed 128. Hunt MD, Pettis GS, McIntosh MA: Promoter and operator determinants for Fur-mediated iron regulation in the bidirectional fepA-fes control region of the Escherichia coli enterobactin gene system. J Bacteriol 1994,176(13):3944–3955.PubMed 129. Escolar L, Perez-Martin J, de Lorenzo V: Coordinated repression in vitro of the divergent fepA-fes promoters of Escherichia coli by the iron uptake regulation (Fur) protein. J Bacteriol 1998,180(9):2579–2582.PubMed 130.

The DNA fragments were separated by agarose (0.8%) gel electrophoresis in TAE buffer (40 mM Tris-acetate, 1 mM EDTA). The gel was stained with ethidium bromide and photographed under UV illumination. Determination

of optimal multiplicity of infection (MOI) Multiplicity of infection is defined as the ratio of virus particles to potential host cells [24]. The titre of prepared phage VX-680 in vivo stock was determined by serial dilution and double-layer plate method. An early log phase of host strain was grown in LB medium at 30°C for 7 h and enumerated by plating PRI-724 nmr samples onto LB agar and then incubated at 30°C for 24 h. Phage stock and hosts were added to LB medium according to six ratios of MIO (0.00001, 0.0001, 0.001, 0.01, 0.1 and 1 PFU/CFU). After 3.5 h of incubation at 30°C, the samples were collected for phage titer determination. One-step growth curve One-step growth curves were performed as described by Leuschner et al. [25] and Pajunen et al. [26]

with some modifications. Briefly, 30 mL of an early-exponential-phase culture (OD650nm = 0.1–0.2) were harvested by centrifugation (10 000 × g, 5 min, 4°C) and resuspended in one-fifth of the initial volume fresh LB medium. Phages were added with an optimal MOI and allowed to adsorb for 10 min at 30°C with the rotary speed of 160 r/min. The suspension was then centrifuged at 12 000 × g for 5 min, resuspended in 30 ml of LB broth and serial dilutions of this suspension were carried out and incubated at 30°C. At regular intervals, aliquots Selleck MRT67307 (100 μL) of each dilution were collected for bacteriophage counts [27]. The burst time and burst size were calculated from the one-step growth curve [18]. Factors affecting phage stability SPTBN5 For investigating pH sensitivity of tested phages, a modified method was used as described by Pringsulaka et al. [1]. 100 μl of phage (about

1010 PFU/ml) was inoculated into a 1.0% Peptone solution with a pH range (pH 4.0, 5.0, 8.0, 9.0, 10.0 and 11.0). The samples were extracted for determining the phage titer after incubating for 60 min. Method used to determining the phage thermal stability was followed as Lu et al. [17]. A 900 μL of 1.0% Peptone solution was preheated to the designated temperature ranging from 50 to 90°C. 100 μl of phage suspension (about 1010 PFU/ml) was added. At regular intervals, the phage titer was determined during 60-min culture. 2KGA production in laboratory scale All fermentations were carried out in 500 mL Erlenmeyer flask containing 40 mL of fermentation medium. 10% (v/v) of seed culture was inoculated and fermented for 72 h at 30°C with a rotatory speed of 270 rpm on rotary shaker. For infected fermentations, 1 mL (108 pfu/mL) of the purified phage was inoculated into the culture after 0 h, 4 h and 8 h of 2KGA fermentation. The fermentation ended until the glucose was consumed to about 0 g/L. As for the experiment of feeding seed culture to the infected 2KGA fermentation, 7.

Discussion Cytotoxicity of haemolytic Listeria spp. in ciliates and amoebae was originally demonstrated by Chau Ly and Müller [7]. They have shown that haemolytic L. monocytogenes and L. seeligeri induce lysis of T. pyriformis and Acanthamoeba castellani during 8-15 days while only few protozoa underwent lysis in the presence of non-haemolytic L. innocua. Our results demonstrated that a L. monocytogenes mutant strain deficient in L. monocytogenes haemolysin, listeriolysin O (LLO) was incapable of impairing T. pyriformis growth compared to the isogenic wild type strain. A saprophytic species of L. innocua expressing LLO acquired toxicity in protozoa and caused their mortality and encystment. Thus, obtained results

suggested that it is LLO that is responsible for L. monocytogenes cytotoxicity in protozoa. Another observed GSK872 LLO activity was stimulation of T. pyriformis encystment. Both cell death and encystment were responsible for decrease of trophozoite counts in the presence of L. monocytogenes. Here our results were in contradiction with previously published [7]. Although cited above authors found that L. monocytogenes accelerates encystment of A. castellani, they did not observe T. pyriformis encystment independently

on bacterial presence [7]. This contradiction is related to the protozoan ability to encyst rather than LLO activity and might be due to different LY2874455 cost sources of a protozoan culture. Cyst GDC-0941 cell line formation by ciliates was described earlier [21] and cysts that we observed for the used T. pyriformis culture were similar to cysts depicted there (see Figure 1). In contrast to wild type L. monocytogenes, LLO-expressing L.

innocua caused a rapid decrease in counts not only trophozoites but as well cysts (see Figure 5). The constitutive LLO expression driven by PrfA* protein, which gene was inserted into the pHly/PrfA* plasmid, might be responsible for higher toxicity of L. innocua transformed with the plasmid. Wild Inositol oxygenase type PrfA protein activity is regulated by co-factor binding, while the PrfA* protein is locked in the active conformation by a Gly145Ser substitution [19]. Obtained results suggested that PrfA activity and LLO expression by intracellular L. monocytogenes might be switch off after host cell encystment but this is not possible for PrfA* protein. Further studies with using L. monocytogenes prfA* [19] are needed to get evidences in support of this suggestion. Another pathogenic bacterium, a common representative of natural ecosystems, L. pneumophila was demonstrated to be cytotoxic for amoeba and to kill A. polyphaga via induction of necrosis due to Legionella pneumophila pore-forming activity [25]. A similar mechanism might be responsible for the cytotoxic effect of LLO. LLO belongs to the family of cholesterol-dependent haemolysins, which includes streptolysin O and pneumolysin O [13, 14]. Proteins of this family can form oligomeric rings that plunge into membrane and generate pores [26].